1. Field of the Invention
The present invention relates generally to the fields of enzymology and carbohydrate chemistry. More specifically, the present invention relates to purified, homogeneous liver GalNAc-1-P kinase and uses thereof.
2. Description of the Related Art
N-acetylgalactosamine (GalNAc) is an important sugar in complex carbohydrates because it is usually the sugar that links the carbohydrate chains to protein in mucins (1-3) and other O-linked oligosaccharides (4-6). In these glycoproteins, the linkage usually involves a GalNAc to serine or threonine attachment site. GalNAc is also found in many of the glycosphingolipids that are present in animal cell membranes (7,8), and is a major component of some proteoglycans such as chondroitin sulfates (9).
The current dogma on the formation and activation of GalNAc in animal cells is as follows (10):
1. Fructose-6-P+Glutaminexe2x86x92Glucosamine-6-P+glutamate
2. Glucosamine-6-P+Acetyl-CoAxe2x86x92GlcNAc-6-P+CoASH
3. GlcNAc-6-P⇄GlcNAc-1-P
4. GlcNAc-1-P+UTP⇄UDP-GlcNAc+PPi
5. UDP-GlcNAc⇄UDP-GalNAc
The enzyme UDP-GlcNAc pyrophosphorylase was first partially purified from calf liver and from Staphylococcus aureus by Strominger and Smith, and various properties of the enzyme were determined. Those enzyme preparations utilized UDP-GalNAc as a substrate at about 2.8% of the rate of the phosphorolysis with UDP-GlcNAc, but it was not clear whether that activity was due to a contaminating pyrophosphorylase. With the partially purified enzyme, the rate of UDP-glucose pyrophosphorolysis was about 30% of the rate with UDP-GlcNAc. The pyrophosphorylase was also partially purified from calf brain and that enzyme also utilized UDP-glucose at about 36% of the rate with UDP-GlcNAc.
The prior art is deficient in the lack of a purification of the enzyme N-acetylgalactosamine kinase to homogeneity for specific uses. The present invention fulfills this longstanding need and desire in the art.
The present invention has demonstrated the presence in pig kidney and some other pig tissues of a new enzyme that phosphorylates free GalNAc in the 1-position to produce GalNAc-1-P. This enzyme has been extensively purified from the cytosol of kidney. The purified enzyme phosphorylates GalNAc, but not galactose, galactosamine, glucose, GlcNAc or glucosamine. This enzyme may represent part of a salvage pathway that allows the organism to reutilize the free GalNAc that arises from the turnover of O-linked glycoproteins and other complex carbohydrates.
In this regard, a UDP-HexNAc pyrophosphorylase from pig liver that catalyzes the formation of either UDP-GlcNAc from UTP and GlcNAc-1-P, or UDP-GalNAc from UTP and GalNAc-1-P (11) was recently purified. Thus, some pig tissues appear to have the necessary enzymes to convert free GalNAc to its xe2x80x9cactivatedxe2x80x9d form, i.e., UDP-GalNAc, for polymerization, without the intervention of UDP-GlcNAc.
In one embodiment of the present invention, there is provided an enzyme N-acetylgalactosamine kinase in isolated and purified form.
In another embodiment of the present invention, there is provided an enzyme that has a molecular weight of about 50 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, has an optimal pH of from about pH 8.5 to about 9.0, and wherein said enzyme catalyzes the phosphorylation of N-acetylgalactosamine and does not phosphorylate N-acetylglucosamine, N-acetylmannosamine, glucose, galactose, mannose, galactosamine and glucosamine.
In another embodiment of the present invention, there is provided polyclonal antiserum recognizing the enzyme of the present invention.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.